The present invention relates generally to devices and methods for performing separation of biological macromolecules (electrophoresis or gel chromatography) and transfer (electro-blot, capillary blot or other means of blotting). The present invention particularly relates to a device, which can perform separation and blotting of protein and nucleic acid samples within one capillary element.
Separation of macromolecules such as proteins and nucleic acids is a necessary step in numerous applications of protein and DNA-RNA analyses in multiple biological, scientific, medical, and forensic applications. The most prominent and widely used techniques for separating macromolecules are chromatography and gel electrophoresis. Following separation by said principles, proteins and nucleic acids are then generally collected in separate volumes or fixed (blotted) to special chemical compounds, mostly nitrocellulose or nylon (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology, Ed. Current Protocols, Wiley, N.Y., 1994), for further processing and analyses. Separation and blotting must be performed independently in time and space, otherwise highly chemically reactive blotting membrane may be contaminated with separated molecules. For this reason after separation is performed, the gel with separated molecules is placed in contact with blotting membrane by mechanical transfer.
Electrophoresis, used for separation, utilizes a physical phenomenon of charged particles"" ability to migrate toward the pole possessing the charge opposite to that of a particle, when it is suspended between opposite poles in an electric field. Conventional gel electrophoresis utilizes a gel slab assembly. Pore matrix of a gel slab is usually made of agarose or polyacrialamide. Pores, which retain macromolecules depending on their physical properties (size, hydrodynamic radius, weight, composition, electrical charge, etc.), form an interacting or sieving matrix. Wells are formed in the upper part of the gel slab during casting procedure. A gel slab assembly is placed into a device for electrophoresis. Samples are then introduced in a band within wells; and an electric field is applied across the slab. The upper and the lower portions of the gel are submerged into separate buffer solution reservoirs. The electric field forces macromolecules of the samples to migrate through the gel. During migration, the macromolecules are xe2x80x9csievedxe2x80x9d by their properties, most often their molecular weight. Specific species of macromolecules will be in bands arranged from top to bottom of the gel (Andrews, A. T. Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Clarendon Press-Oxford (1986), 2nd Ed).
In the process of DNA separation, the DNA fragments (DNA ladders) can be labeled before or after separation with either radioactive or florescence labels (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology. Current Protocols, Wiley, N.Y., 1994, Chapter 2). In DNA sequencing procedures, each of the four types of a nucleotide can be labeled with specific probe, which will appear at termination of DNA fragment. A mixture of different reactions can be electrophorized and separated according to the label from one reservoir in the gel.
After separation is complete, deposition membrane with blotting material is placed in contact with gel. A common transfer process is called xe2x80x9celectro-blotxe2x80x9d transfer. In the xe2x80x9celectro-blotxe2x80x9d transfer process, the macromolecules in the gel slab move under an electric field to a blotting membrane. In designing an electro-blot transfer system it is essential that the blotting membrane be in close contact with the gel slab. Presence of gas bubbles between the gel slab and blotting membrane will prevent the band images from being transferred properly. It is also important to maintain a uniform electric field across the electro-blot sandwich. Transfer of the gel slab onto the nitrocellulose membrane must be carefully performed so that the macromolecules on the gel membrane are not removed or contaminated. After transfer, a labeling procedure must be employed, and a detection technique must be utilized so that the samples can be analyzed. A commonly used detection method involves staining and de-staining of the gel slab. This technique imposes staining of the entire gel with a dye that only adheres to the macromolecules. Then a de-staining process is performed, wherein dye not adhered to the macromolecules is washed away; bands of macromolecules thus become detectable. Another common detection method is the use of antibodies. Bands of proteins or samples are blotted or transferred to a binding membrane, which binds macromolecules. Then, a known antibody is introduced. The antibody combines with a specific protein if it is present in a sample. In order to detect the antibody-protein combination, the antibodies are labeled with fluorescent or radioactive tags or have enzyme activity, which is further detected by separate methods (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology, Ed. Current Protocols, Wiley, N.Y., 1994, Chapter 10).
Capillary electrophoresis offers some advantages not available with other separation methods, such as slab gel separation, HPLC, or column chromatography (Krylov, S. N., and Dovichi, N. J. Capillary electrophoresis for the analyses of biopolymers. Analytical Chemistry, 2000, Vol. 72, No. 12:111R-128R). The major advantage of the capillary electrophoresis is the speed of the analysis (few minutes, compared to few hours by other methods). Highly efficient dissipation of electro-resistive generated heat in the capillary is provided by a large surface to volume ratio. Decoupling of gel temperature from electro-resistive generated energy provides greater effective field strength. Migration rate varies directly with the field strength over a linear range, which can be extended using gel-filled capillaries. Thus, separations at higher fields can be performed with reduced running time. The advantage of a single capillary can be further extended by use of an array of coupled capillaries with equal characteristics. A possibility to process them together under identical conditions allows technology for protein separation and DNA sequencing (Dolnik V. DNA sequencing by capillary electrophoresis. J Biochem. and Biophys. Methods. 1999, V.41, No. 2-3:103-119).
Collection of samples in capillary electrophoresis poses, however, substantial technical problems (Altria, K. D. Overview of capillary electrophoresis and capillary chromatography. J. Chromatography, 1999, Vol. 286, No 1-2:443-63; Swinney, K., Bornhop, D. J. Detection in capillary electrophoresis. Electrophoresis, 2000, Vol. 21, no. 7:1239-50). Samples are eluted from the capillary at a certain point by either pressurized flow or electroelution. Method for sample collection by electroelution employs standard capillary electrophoretic equipment. Several parameters of the system must be precisely controlled (velocity of migration of a sample, distance between the detection point and the end of the capillary, etc). To collect fractions in appropriate vials, capillaries, etc, one must know the exact time, when a zone appears at the exit end of the capillary, as the variation of migration rates in capillary electrophoresis can be more than 2%. The time necessary for the zone of interest to move through the distance between the detection point and the capillary exit is calculated after detection of the zone of interest is done. Electric current is then turned off, and the capillary is removed from the apparatus and placed into a collection vial. Current is applied for a predetermined time so that the zone migrates into the collection reservoir with buffer from the capillary. Pressure can also be applied to remove the sample from the capillary. After collection of the zone is accomplished, the capillary is placed back into the electrophoretic device, and separation continues. A set of collection reservoirs, or capillaries, containing a collection buffer and electrode, can be positioned close to the exit end of the capillary; collection of samples can be performed, while the electric current is temporarily turned off.
Another approach for collecting samples from a capillary is positioning samples on a membrane. According to the technique, the exit end of the capillary is in contact with a wet surface of a moving blotting membrane, which also serves as an electrode. Separated proteins, or DNA fragments, are transferred to deposition membranes either by electrophoresing the bands onto a moving deposition membrane (direct electroblotting) or by various elution techniques implying gradient of hydrostatic pressure, capillary forces, and others. Standard direct blotting devices use a deposition membrane attached to a conveyor belt in the lower buffer chamber that remains totally submerged during electrophoresis. These devices require physical removal of the nylon or other membrane from the conveyor belt. Submersion of the deposition membrane makes detection and processing a difficult task.
Prior art:
U.S. Pat. No. 4,622,124 discloses a device for horizontal electroblotting of electrophoretically separated material. A liquid tight container is provided having a support assembly, horizontally disposed in a chamber defined by the container. Electrodes are located below and above the support assembly, and a barrier is provided to prevent bubble attachment and uneven blotting. U.S. Pat. No. 4,589,965 presents a method for electroblotting, whereby an electrophoretically resolved material in a gelatin sheet is transferred to a membrane. The gelatin sheet is in contact with an immobilizing material, sandwiched between two plate electrodes. U.S. Pat. No. 4,994,166 to Fernwood et al describes a single apparatus for slab gel electrophoresis and blotting, both of which are performed in a single tank cell, which contains separation electrodes along opposing vertical walls and blotting electrodes arranged horizontally above and below the level of gel placement. The cell is operated in separatory and blotting modes, in which separatory and blotting electrodes are separately energized. No means for separation of gel and blotting membrane are provided.
U.S. Pat. No. 4,812,216 to Hurd explain a method and apparatus for supporting and handling blot membranes during the course of blotting, analysis, and storage. U.S. Pat. No. 5,039,493 to Oprandy describes a positive pressure blotting apparatus having a bottom section, a middle section, and a top section. The first volume of space is encompassed between the top section and the middle section. Positive pressure is maintained in the first volume of the apparatus. A hydrophobic filter means for binding biological materials is positioned on, or in, the middle section, and a means is provided to secure the top, middle and bottom sections together to form a pressure blotting apparatus. U.S. Pat. No. 5,149,408 to Perlman shows a capillary blotting sandwich for transferring soluble macromolecules in a liquid medium from a liquid-permeable matrix to a semi-permeable receiving membrane. The blotting sandwich includes: (i) a liquid-permeable matrix layer comprising soluble macromolecules, said matrix having at least one flat surface; (ii) a semi-permeable membrane sheet layer disposed on said flat surface; (iii) an interlocked hydrophilic absorbent fiber-containing material in the form of a non-woven absorbent felt sheet, which facilitates capillary transport of a solution through said membrane.
U.S. Pat. No. 5,013,420 to Schuette presents electrophoresis-blot transfer apparatus, in which a buffer tank is common to both the electrophoresis and electro-blot transfer. Combination of two procedures (separation and transfer) requires a two-step operation with reassembly of an electro-blot sandwich. U.S. Pat. No. 4,889,606 to Dyson et al describes a method and apparatus for use in electrophoresis and blotting, in which electrophoresis is carried out on a gel, supported by a rigid porous plate. The gel is cast onto the porous plate with a transfer membrane between the porous plate and the gel. The method consists of the first stage, using electric field produced by electrodes to draw the samples through the gel; and the second stage, drawing the distributed macromolecules onto the transfer membrane. Since separating gel and transfer membrane are not separated by a barrier, contamination of the blotting membrane occurs while separation. U.S. Pat. No. 4,589,965 to Kreisher describes a method for electroblotting, whereby an electrophoretically resolved material in a gelatin sheet is quickly and efficiently transferred to a membrane with high pattern definition and resolution.
U.S. Pat. No. 5,593,561 to Cognard and Hache shows a multiple electrophoresis method for controlled migration of macromolecules and transfer thereof to the membrane in a vessel, containing a plurality of parallel elongate electrodes. The first electric field, established between electrodes, provides means for macromolecular separation in a gel, and the second electric field, perpendicular to the first, provides means for transferring the macromolecules onto the membranes. In the described method, at first, electrodes and transfer membranes are assembled in the vessel, which is then filled with gel. After the separation of macromolecules in a gel and transfer to membranes, gel is liquefied, dissolved, or decomposed, allowing the removal of membranes. Closely related U.S. Pat. No. 5,102,524 to Dutertre describes a multiple electrophoresis method, where different sets of electrodes are used in a two-step process to first separate macromolecules and then to transfer them to a deposition membrane. No means to separate the deposition membrane from the gel during the first step of electrophoresis are provided. U.S. Pat. Nos. 4,849,078 and 4,911,816, both to Love et al, present apparatus for carrying out horizontal electrophoresis and subsequent vacuum-assisted transportation of macromolecules to a support membrane. Process is performed in two steps: first, electrophoresis is performed; and second, deposit membrane is put in contact with gel, and vacuum transfer is conducted.
U.S. Pat. No. 5,217,592 to Jones describes apparatus, which includes the combination of submarine gel tank for electrophoresis separation with a vacuum applying means, which transfer the separated molecules from the gel into the deposition membrane, to a filter membrane by means of controlled vacuum. No means to separate the deposition membrane from the gel during the first step of electrophoresis are provided. U.S. Pat. No. 5,155,049 to Kauvar teaches a technique for passage of liquid through a membrane putatively containing in its interstices at least one substance, for which detection is desired. Further, it comprises positioning donor and acceptor bibulous matrices onto either surface of the membrane, squeezing the resulting sandwich. This technique permits the application of small volumes of reagents or wash to the membranes and the facile recovery of the waste.
U.S. Pat. No. 5,445,723 to Camacho describes blotting apparatus for transferring electrophoretically separated molecules from the gel into a transfer stack. Apparatus includes a resilient anode surface for transfer of molecules, which is mounted on a mechanical carrier arm, which moves the anode surface over the membrane during transferxe2x80x94mechanically moved device.
U.S. Pat. No. 5,234,559 to Collier et al explains an apparatus for direct blotting and automated electrophoresis, transfer, and detection of biomolecules. Separation and transfer module consists of a separating gel and transfer membrane, stabilized by a frame, which is moved mechanically in orthogonal direction to the gel. Moving frame with the membrane is in contact with the edge of the gel, towards which separated fragments are driven electrophoretically.
U.S. Pat. No. 5,279,721 to Schmid describes an automated electrophoresis and transfer apparatus for separating macromolecules and blotting them to a transfer membrane, which includes a housing having a perforated intermediate horizontal partition defining an upper reservoir and a lower chamber. A transfer membrane is positioned over the partition; an impermeable sheet is interposed between the membrane and the gel member and withdrawn prior to transfer operation. Cooperation with the impermeable sheet substantially prevents chemical solutions from permeating the transfer membrane prior to transfer operation. In this embodiment, subdivision of separation and transfer processes is provided by means of mechanically moving an impermeable membrane.
U.S. Pat. Nos. 4,631,120 and 4,631,122, depict apparatus and method for a direct blotting process, which provides one or more collecting surfaces mounted on a conveyer belt or tape and advances the belt so that it slides over the end of the gel to collect separated particles and transport them with the belt away from the gel to a location, where further processing may be performed. Both vertical and horizontal embodiments are disclosed. Contact of this nature is damaging to the surface and may remove separated material. Also apparatus does not allow handling and identification of a multiplicity of samples in sequence. U.S. Pat. No. 5,514,256 to Douthart et al describes a direct blotting electrophoresis unit for DNA separation. The DNA sequence is deposited on a membrane attached to a rotating drum. Separation of DNA is performed in a capillary-like mini-gel system. A rotating drum with a deposition membrane is adjacent to the edge of the gel, and transfer of molecules to the gel occurs by means of electrophoretic transfer.
Claimed apparatuses and methods possess substantial disadvantages: after separation is completed, direct blotting in combination with slab gel electrophoresis requires mechanical means for changing the configuration of the system in order to put the blotting element in contact with separating gel. In capillary electrophoresis system, direct blotting appears to be impossible because a very thin layer of interactive matrix is in a very close proximity to the walls.
Use of semipermeable membranes for assisting the capillary separation has been claimed previously, though for different purposes. For example, U.S. Pat. No. 5,985,121 to Wu et al describes apparatus for capillary electrophoresis carrying out on-line sample preparation by means of a semipermeable membranes connected to the capillary separation column for selective introduction of ampholytes into the capillary separation column. Transfer procedures are not described in this invention. U.S. Pat. No. 5,338,427 to Shartle teaches a disposable cartridge for a capillary electrophoresis instrument, in which short capillary tube segments are used for simultaneous multiple lane separations. The cartridge contains all separation components of the instrument, which come in contact with the sample and is capable of automatically loading a quantitative portion of a bulk sample into the capillary tube segments. Electrophoresis occurs without bulk flow through the capillaries, which are scanned in situ by the instrument. U.S. Pat. No. 5,169,511 depicts capillary tube for electrophoresis with a sample cup, consisting of two wells, bottoms of which are covered with semipermeable membrane. Membrane allows the flow of buffer, but not the migration of separated sample. U.S. Pat. No. 5,482,613 describes a method for making a gel plate with a microporous membrane by means of rectangular frame cassette and means of tensioning a membrane deposited on a frame. U.S. Pat. No. 4,512,896 to Gershoni teaches a method of transfer of macromolecules to a mobilizing matrix, which is a charge modified microporous membrane. U.S. Pat. No. 5,897,817 shows a process for making medical device from a cellulose acetate hollow fiber semipermeable membrane. In this process, a molten liquid, comprising cellulose acetate, is extruded to produce a membrane. The solvent and the non-solvent are removed from the membrane to produce a semipermeable membrane, having water permeability. The semipermeable membrane can be incorporated in casing in order to produce a product. U.S. Pat. No. 5,131,994 to Shmidt and Cheh explains a method and apparatus for affecting an electrophoretic separation of charged particles. Fractionation chamber comprising a semipermeable membrane is used, wherein a trans-membrane force holds the particles to be separated against the membrane surface, while a voltage gradient is applied to separate particles. U.S. Pat. No. 4,964,961 to Brautigam and Gorman describes apparatus for electroelution of components, separated by preparative electrophoresis on a gel comprising a means for separation of molecules and elution of a selected fraction through a side of separation vessels using a dialysis membrane.
U.S. Pat. No. 5,840,169 to Andersen presents an apparatus and process for electroelution of a gel, comprising of a device with parallel adjacent chambers. Gel with separated macromolecules is placed on top of the apparatus; and electroelution occurs through a semipermeable membrane, placed on the other open sides of the chambers. Macromolecules in the gel migrate into the elution buffers in the chambers. Apparatus and method do not provide means for blotting.
U.S. Pat. No. 5,284,559 to Lim and Hixton describes device for electrophoresis comprising of a pair of spaced apart plates with a separating gel in between and a semipermeable membrane connected to the end of the plates. Membrane forms a collection channel for receiving molecules, which have traveled through the gel, and allows performance of fraction collection. Means for blotting are not disclosed. U.S. Pat. No. 5,427,664 to Stoev demonstrate a free solution electrophones-membrane-filter trapping assay apparatus, which includes a container and at least one porous membrane. The porous membrane and container define a chamber capable of holding run solution. A specimen containing particles to be classified is juxtaposed with the run solution. Particles, which are smaller in size than the pores of the membrane, pass through the membrane, while particles having a larger size do not. A method of classifying particles includes steps providing a sample of particles and a porous membrane, positioning a run solution between the sample and the membrane, and applying an initial voltage across the sample, run solution and membrane.
U.S. Pat. Nos. 4,992,172 and 5,160,626, both to Pemawansa et al, are examples of various membrane compositions, including the ones, which use microporous composites for direct blotting. U.S. Pat. No. 4,992,172 describes a blotting composition, comprising a substrate applied to an activated microporous membrane comprising a membrane polymer and having (1) an internal surface comprising interstices or pore surfaces and (2) an external surface; wherein substantially all said surfaces of the microporous membrane are coated without significant chemical reaction to substantially all said available surface areas of said microporous membrane and thereby activated by a polyaldehyde-containing mixture in an amount sufficient to effectively activate the microporous membrane; and wherein the mixture is soluble enough to penetrate substantially all said interstices of the microporous membrane without substantial damage to the pore structure. Means of separation and/or modification of membranes in the process of transfer are not claimed. U.S. Pat. No. 5,160,626 describes a method of transferring a biological sample to an immobilizing matrix, comprising applying the sample to an activated microporous membrane having: 1) an internal surface comprising interstices or pore surfaces; and 2) an external surface, which membrane comprises a membrane polymer that is non-covalently activated by having all said surfaces of the membrane physically coated with a polyaldehyde mixture in an amount sufficient to provide effective free aldehyde functionality on said surfaces for covalent bonding between the thus coated membrane and aldehyde reactive material of said sample and to thereby non-covalently activate the microporous membrane for said bonding without substantial damage to the pore structure. Means of combined separation and blotting are not disclosed.
The major disadvantage of the prior art is that the methods and apparatuses described allow performing only one of the two processes at a time and require a mechanical rearrangement of the geometry of the system to combine blotting and separating elements. Two separate units increase cost, labor and time of the procedure, which is a significant disadvantage with conventional devices. Mechanically based devices are complex and expensive; they cannot be used as disposables. In a standard setup for capillary separation molecules of interest, if direct blotting to the walls of the capillary is attempted, the molecules of interest will be attached to the walls along the whole length of the capillary, making distinction impossible. For that reason, the entire wall of the capillary element must be inert and should not allow any interaction between walls and separated molecules. Thus, detection of capillary electrophoresis or chromatography was previously possible only at the exit end of the capillary. The method of the instant invention allows modification of chemical properties of the walls of separating element, so that walls are transformed from impermeable to permeable state after separation by means of chemical or physical modification and thus allow a free passage of separated molecules towards the blotting element through the walls of the capillary. Previously described capillary-based separation techniques do not allow to perform separation and direct blotting, as they require detection or blotting only after separated materials leave the capillary. In formerly described capillary separation devices, these processes cannot be combined together in one capillary element. The capillary electrophoresis system and method of the instant invention have solved these problems of prior art systems.
It is an object of the present invention to provide an apparatus that positions a transfer membrane (blotting membrane) relative to a separating element (sieving or interacting matrix such as gel) by means of permeable membrane, whose properties can be chemically or physically modified, and when the gradient of driving forces is changed from the direction of separation to direction favorable to transfer, the transfer to the membrane is conducted in a time-controlled fashion. It is a further object of the present invention to combine separating element and blotting element in one composite to eliminate the necessity to perform two separate processesxe2x80x94separation and blotting. A feature of the present invention is the means to provide a combined apparatus for separation and blotting in one capillary-based unit with means of direct blotting to the walls of a disposable capillary. It is yet another advantage of the present invention, that it allows to use very long capillary elements, where length to diameter ratio of capillary can be more than a 100, thus providing a high resolution of separation. It is a further advantage of the present invention that the apparatus and method disclosed are amenable to automated process and allow to provide uniform, repeatable and well-controlled conditions for performing separation and blotting even in non-specialized environment (field environment). These and other objects, features and advantages of the invention will become apparent upon having reference to the following description of the preferred embodiments. The present invention meets the above objectives and provides advantages theretofore unavailable in conventional devices.
The invention is directed to a system for separation of macromolecules according to their dimensions and/or charge (capillary or blot electrophoresis or gel chromatography) and method for precise post-separation blotting of said molecules to the accepting element. A disposable separation element (capillary) contains inside a sieving or interaction matrix; an external layer of blotting material, positioned close to the boundary of said sieving or interaction matrix; and/or a membrane with changeable permeability for separated material, which separates blotting layer from the sieving or interaction matrix. After separation of macromolecules in capillary with initially non-permeable walls, physical of chemical modification of the intermediate membrane is performed, which is followed by change in the vector of driving forces for transfer, so that separated molecules are moved through the walls of the capillary and blotted to the outer layer of blotting material. The system of the invention includes a disposable separation element (capillary), which can be stretched or coiled inside reservoir; an electrophoresis buffer reservoir to supply electrophoresis buffer to the entrance and exit of the capillary; reservoirs to supply buffer; and modifying solution to a blotting material positioned as an outer layer at the body of capillary element. Means of modification of separation membrane include, but not limited to, chemical or physical modification to change permeability of the membrane for separated material after accomplishment of the separation in said sieving or interaction matrix. Change in driving force for the separation material may include, but is not limited to electrical charge application, bulk flow of fluid, hydrostatic pressure, or gravity.
Post-blotting processing of the sample components in the said system can include any type of analysis, reaction, modification, collection, or purification. For example, the blotting element may be used to transfer the separated and blotted sample components to analytical devices and/or further processed in antigen-antibody reaction or hybridized with DNA or RNA probes. Also blotted samples may be purified or processed by radioactive analysis or electrochemical analyses.